Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2016-06-27 to 2016-08-26 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

The mono-ester fraction of glycerol and the mono-ester fraction of pentaerythritol are UVCBs, made up of a range of mono-ester constituents varying based on the resin acid component. The resin acid composition is dependent on the composition of the rosin starting material, but is not affected by the alcohol that the rosin is reacted with (glycerol or pentaerythritol). Therefore, although there is variability in the resin acid composition, this is the same for both glycerol and pentaerythritol esters and is not considered to affect the read across potential. The Final Decision letters for both substances state that “There is considerable structural similarity between the ester compounds and therefore a read-across within the whole fraction of each level of esterification using selected constituents as source substances is considered appropriate”.

Mono-ester constituents of both substances consist of a resin acid, joined to an alcohol by an ester bond. Based on QSAR predictions, the identity of the resin acids is not considered to have a significant impact on biodegradation potential. Both substances contain both hydrogenated and non-hydrogenated resin acids, and the proportion of hydrogenated constituents is not likely to vary significantly between the two mono-ester fractions. Therefore, the only structural difference between glycerol and pentaerythritol mono-esters is the identity of the alcohol. Glycerol mono-ester constituents contain two hydroxyl groups, and pentaerythritol mono-ester constituents contain three.

When mono-ester constituents are degraded, the first step in the degradation process is likely to be breaking of the ester bond. This bond is the same for both glycerol and pentaerythritol mono-esters, and the addition of an additional hydroxyl group to the pentaerythritol mono-ester would not affect the degradation of this bond. The additional functional group does not increase steric hindrance and thus would not limit the rate of degradation for these constituents. Primary degradation of mono-ester constituents is therefore considered to be equivalent for both glycerol and pentaerythritol mono-esters.

The degradation products from the expected primary degradation of both mono-ester fractions are resin acids and glycerol or resin acids and pentaerythritol. The resin acids would be the same for both mono-ester fractions. The other degradation product would be different but, as both glycerol and pentaerythritol are readily biodegradable, the alcohol is not expected to affect the ultimate degradation potential of the test item.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source: Mono-ester fraction of Resin acids and rosin acids, hydrogenated, esters with glycerol
Target: Mono-ester fraction of Resin acids and rosin acids, hydrogenated, esters with pentaerythritol

3. ANALOGUE APPROACH JUSTIFICATION

Degradation of both glycerol and pentaerythritol mono-esters is expected to occur initially through breaking of the ester bond joining the resin acid to the alcohol, and this is supported by predictions made using the EAWAG database (EAWAG Aquatic Research Biocatalysis/Biodegradation Database), which assesses microbial degradation, as outlined in the Final Decision letters for each substance. This degradation process is expected to occur at a similar rate for both substances as the identity of the alcohol is not expected to affect degradation of the ester bond. Although the alcohol is different, in both cases only one resin acid is joined to the alcohol and therefore the ester bond is equally accessible for microbial degradation in all mono-ester constituents.

The rate and extent of primary degradation is expected to be similar between the two substances, and one of the primary degradation products (resin acids) will be identical for both mono-ester fractions. The only difference in primary degradation products will be the alcohol, glycerol or pentaerythritol, but as both are readily biodegradable the identity of the alcohol is not expected to affect the biodegradation potential of these constituents.

Neither glycerol nor pentaerythritol mono-ester constituents are considered to be toxic to microorganisms. Toxicity controls included in ready biodegradation studies conducted with Resin acids and rosin acids, hydrogenated, esters with glycerol and Resin acids and rosin acids, hydrogenated, esters with pentaerythritol (where the test items would have contained mono-ester constituents), did not indicate any toxicity to microorganisms used in the studies (Inveresk Research 2002).

The toxicity control included in the source study with mono-ester constituents of Resin acids and rosin acids, hydrogenated, esters with glycerol also did not show any toxicity to microorganisms. There is no reason to believe, based on the structure of the pentaerythritol mono-ester constituents, that they would be more toxic to microorganisms. Potential toxicity to microorganisms is therefore not expected to affect the read across potential between the two mono-ester fractions.

Degradation can be affected by bioavailability of the test item, which depends on solubility, volatility and the potential for adsorption of the test item. As the constituents of concern are not available as isolated constituents, test data for these properties is not available. However, QSAR predictions have been run using the EPIWIN models (see full read across justification for more details).
Both mono-esters have very low volatility (vapour pressure 1.74E-11 to 5.27E-11 mm Hg for glycerol mono-esters; 1.92E-14 to 5.73E-14 mm Hg for pentaerythritol mono-esters) and therefore losses due to volatilisation are not expected for the mono-ester fraction of either UVCB substance.

Both glycerol and pentaerythritol mono-esters have limited solubility, (water solubility 0.269 to 2.85 mg/L for glycerol mono-esters; 0.812 to 8.77 mg/L for pentaerythritol mono-esters). As the glycerol mono-ester test item was poorly soluble, this was accounted for in the design of the ready biodegradation study, with the substance being initially dissolved in acetone and applied to the walls of the test vessel. At the start of the test a film of test item was then observed to float in the test vessels. This method of introducing the test item is considered to increase the availability of the test item to microorganisms in the test system and therefore the potential for degradation. The same technique would be used if the pentaerythritol mono-ester were tested and therefore the slight difference in water solubility between the two mono-esters is not considered to impact on the potential to read across from the glycerol to the pentaerythritol mono-esters. Based on the QSAR predictions, pentaerythritol mono-esters may be slightly more soluble and therefore, if anything, slightly more bioavailable than glycerol mono-esters.

During a biodegradation study, there is also the potential for the test item to adsorb to the test vessels and therefore decrease the availability of the test item. Based on predicted log Koc values for representative mono-ester constituents (2.86 to 3.31 for glycerol mono-esters and 3.36 to 3.81 for pentaerythritol mono-esters), both have similar potential for adsorption, with the Koc values for pentaerythritol mono-esters slightly higher than for glycerol. Glycerol mono-esters may therefore be slightly less likely to adsorb during a biodegradation study, and may therefore have greater potential for biodegradation. However, any difference in adsorption potential between the two is likely to be limited and is not considered to have a major impact on biodegradation potential.

When QSAR predictions for biodegradation are assessed (based on BIOWIN models 5 and 6 as the models most applicable to these structures), glycerol mono-ester structures are ‘readily biodegradable’ based on BIOWIN 5 predictions and ‘not readily biodegradable’ based on BIOWIN 6 results. Pentaerythritol mono-esters are ‘readily biodegradable’ based on BIOWIN 5 predictions and ‘readily biodegradable’ for some mono-ester constituents based on BIOWIN 6, with other constituents close to being considered ‘readily biodegradable’ (BIOWIN 6 predictions 0.448 to 0.646, readily biodegradable if >0.5).

The higher predicted biodegradation potential for pentaerythritol mono-ester constituents is likely to be due to the additional OH group, leading to slightly higher water solubility and therefore biodegradation potential. Based on these BIOWIN predictions, pentaerythritol mono-ester constituents may have more potential for biodegradation and therefore testing the glycerol mono-ester fraction and reading the results across to pentaerythritol mono-esters could be considered a worst-case approach. However, as the structural difference between glycerol and pentaerythritol ester constituents is limited to one additional OH group for the pentaerythritol mono-esters, any difference in ionisation potential which may affect solubility and degradation potential is likely to be limited.

In a Proposal for Amendment to the Draft Decision Letter, the Netherlands stated that ‘as pentaerythritol has a larger structure (containing more functional groups) compared to glycerol, we expect that HRPE will be more persistent and bioaccumulative compared to HRGE’. Although the structure of pentaerythritol mono-esters is larger, as discussed above the available QSAR results show that both glycerol and pentaerythritol mono-esters are likely to behave in a similar manner in the environment, and in fact testing the glycerol mono-esters could potentially be worst case. Based on these QSAR results, and the fact that a sample with a higher percentage of mono-ester constituents can be prepared for the glycerol mono-ester, testing the glycerol mono-ester and reading across to the pentaerythritol mono-ester is considered to be justified.

4. DATA MATRIX

See full read across justification attached in the robust study summary.
Reason / purpose:
read-across source
Preliminary study:
During preliminary investigations (non-GLP) different methods for test item application were trialled. For the main study, the test item was applied by preparing a stock solution in acetone with a concentration of 26.4 mg/mL. 100 µL of this was pipetted around the wall of the test vessel and the acetone allowed to evaporate over 3 days. It was observed in the preliminary study that when the test item was applied as described, the test item was forming a film on the glass wall surface (approximately two millimetres wide). This film detached from the wall after the test solutions were filled in and was floating in the solution. Therefore, this way of application was considered optimal, because the test item is in good contact with the test solutions.
Test performance:
In the toxicity control, biodegradation of 44% was determined after 14 days and it came to 50% (95% confidence interval: 48 - 52%) after 28 days. The test item in the toxicity control did not inhibit the biodegradation of the reference item.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
34
Sampling time:
28 d
Remarks on result:
other: mineralisation
Remarks:
95% confidence interval: 32 - 36%
Key result
Parameter:
% degradation (CO2 evolution)
Value:
35
Sampling time:
60 d
Remarks on result:
other: mineralisation
Remarks:
95% confidence interval: 32 - 38%
Key result
Parameter:
% degradation (test mat. analysis)
Value:
89
Sampling time:
28 d
Remarks on result:
other: primary degradation (quantified results)
Key result
Parameter:
% degradation (test mat. analysis)
Value:
95
Sampling time:
60 d
Remarks on result:
other: primary degradation (quantified results)
Parameter:
% degradation (test mat. analysis)
Value:
93
Sampling time:
28 d
Remarks on result:
other: primary degradation
Remarks:
relative % degradation based on peak area at Day 28 compared to Day 0, from metabolite analysis report
Details on results:
A decline of resin acids was observed between test start and day 3 of the study and an increase of these acids was observed on day 7 of the study. This is in correlation with the start of the primary degradation (57% degradation) of the mono ester fraction between day 3 and day 7.Primary degradation of the mono ester fraction of dehydroabietic acid was observed to be faster than that of the hydrogenated mono ester fraction of resin acids. An approximate amount of 15% of the mono ester fraction of dehydroabietic acid was still present on day 7, whereas only 35% of the mono ester fraction of the hydrogenated resin acid derivatives was degraded. By day 28, approximately 90% of both mono ester fractions were degraded. The amount of resin acids decreased between day 7 and day 10 and increased again between day 10 and day 21 but on a level lower than that on day 7. The amount decreased again between day 21 and day 28. The maximum amount of dehydroabietic acid was obtained on day 10 but then decreased steadily. Difficulties in fully interpreting the levels of resin acids over the course of the study are due to the concurrent formation and degradation.

The test item is classified as not readily biodegradable when considering the ultimate degradation results. Nevertheless, the test item reveals a considerable potential for biodegradation, as the determination of the primary degradation of the monoester fraction as well as the determination of mineralisation shows.
Results with reference substance:
The percentage degradation of the reference substance exceeded the pass level of 60% within 7 days and came to 82% (95% confidence interval: 80 - 84%) after 28 days.

Table 1. Mean CO2 production and biodegradation

 Day

 Solvent control

 Inoculum control

 Reference substance      

 Test item      

 Toxicity control      

 

 Mean CO2 (mg C/L)

 Mean CO2 production (mg C/L)

 Mean CO2 (mg C/L)

 Mean CO2 production (mg C/L)

 Mean CO2 (mg C/L)

 Net mean CO2 production (mg C/L)  % degradation (95% confidence interval)  Mean CO2 (mg C/L)  Net mean CO2 production (mg C/L)  % degradation (95% confidence interval)  Mean CO2 (mg C/L)  Net mean CO2 production (mg C/L)  % degradation (95% confidence interval)

 0

 0.31

 -

 0.43

 -

 0.43

 -  -  0.25  -  -  0.35  -  -

 3

 0.85

 0.54

 nd

 nd

 nd

 nd  nd  0.92  0.13  1  nd  nd  nd
 7  0.81  0.50  0.95  0.52  14.74  13.79  79  3.71  2.96  12  nd  nd  nd
 10  1.14  0.83  nd  nd  nd  nd  nd  6.16  5.08  21  nd nd nd 
 14  1.15  0.84  1.28  0.85  15.50  14.22  81  7.24  6.15  25  19.72  18.53  44
 21  1.51  1.20  nd  nd  nd  nd  nd  9.25  7.80  32  nd  nd  nd
 28  1.90  1.59  2.60  2.17  16.94  14.34  82 (80 - 84)  10.25  8.41  34 (32 - 36)  23.02  21.08  50 (48 - 52)
 35  2.09  1.78  nd  nd  nd  nd  nd  11.82  9.79  40  nd  nd  nd
 42  1.75  1.44  nd  nd  nd  nd  nd  10.57  8.88  36  nd  nd  nd
 49  1.77  1.46  nd  nd  nd  nd  nd  10.96  9.25  38  nd  nd  nd
 56  1.16  0.85  nd  nd  nd  nd  nd  9.87  8.77  36  nd  nd  nd
 60  1.35  1.04  nd  nd  nd  nd  nd  9.80  8.51  35 (32 - 38)  nd  nd  nd

Mean CO2 values have been corrected for NaOH

nd = not determined

Table 2. Primary degradation of the test item

 Day  Toxicity control     Solvent control     Test item   
   Measured concentration of monoesters (mg/L)  % primary degradation  Measured concentrations of monoesters (mg/L)  % primary degradation  Measured concentrations of monoesters (mg/L)  % primary degradation
 0  24.0, 22.7  -  < LOQ  -  22.6, 20.8  7
 3  nd  nd  nd  nd  23.3, 24.3  0
 7  nd  nd  nd  nd  7.07, 5.20  74
 10  nd  nd  nd  nd  3.33, 3.90  84
 14  nd  nd  nd  nd  4.43, 3.66  82
 21  nd  nd  nd  nd  3.66, 2.36  87
 28  nd  nd  nd  nd  2.15, 3.22  89
 60  nd  nd  nd  nd  1.49, 1.26  94

Measured concentrations reported as replicates

nd = not determined

Table 3. Metabolite identification by GC-MS (taken from the metabolite identification report)

 Group  Day                  
   0  3  7  10  14  21  28
 Area - Group 1 (hydrogenated resin acids)  132523  75118  149314  27568  46132  49775  27585
 Area - Group 3 (dehydroabietic acid)  185282  11046  27738  24199  8193  7323  5317
 Area - Sum of Groups 1 and 3  317805  86164  177052  51767  54325  57098  32902
 % test item - Sum of Groups 1 and 3  3.56  0.97  1.99  0.58  0.61  0.64  0.37
               
 Area - Group 2 (monoester fraction of hydrogenated resin acids)  5244629  5651691  3377386  2705210  2005680  1658100  550585
 Area - Group 4 (mono ester fraction of dehydroabietic acid)  3674796  3818124  432561  493826  113708  60841  43329
 Area - Sum of Groups 2 and 4  8919425  9469815  3809947  3199036  2119388  1718941  593914
 % test item - Sum of Groups 2 and 4  100  106  43  36  24  19  7
Validity criteria fulfilled:
yes
Remarks:
Degradation of the reference item must be > 60% by day 14 (actual 81%) and the maximum amount of TIC in the inoculum controls must be < 3 mg C/L at the end of the test (actual 2.60 mg C/L)..
Interpretation of results:
not readily biodegradable
Remarks:
Nevertheless, the test item has a considerable potential for biodegradation, demonstrated by the primary degradation of the monoester fraction.
Conclusions:
The ultimate biodegradation of the test item was 34% after 28 days and 35% after 60 days, based on CO2 production. Primary biodegradation of the monoester fraction reached 74% after 7 days, 89% after 28 days and 95% after 60 days. Hydrogenated resin acids and dehydroabietic acid are early metabolites in the primary degradation of the mono ester fraction of the test item.
Executive summary:

The ready biodegradability of the test item Monoesters of hydrogenated rosin with glycerol was determined with a mixed inoculum of the aqueous phase of non-adapted activated sludge and pre-treated, non-adapted standard soil in the Headspace Test following OECD 310 (Noack Laboratorien 2017a). Biodegradation was assessed after 28 days, and the test was then extended to 60 days. Mineralisation of the test item was followed by TIC analyses to assess CO2 production. Primary degradation of the monoester fraction was determined by GC-MS analysis of representative monoester constituents.

The study was conducted using a nominal test item concentration of 33 mg/L, corresponding to a carbon content of 24.6 mg C/L. A stock solution of the test item in acetone was prepared and introduced to the test vessel by pipetting the stock solution around the wall of the test vessel to produce a big surface area. The acetone was allowed to evaporate over 3 days before the test start. CO2 was captured by sodium hydroxide solution and carbon concentrations were determined with a carbon analyser. The test included a reference item control, toxicity control, solvent control and inoculum control. All validity criteria for the study were met. This study is considered to be reliable without restriction (Klimisch 1).

The ultimate biodegradation of the test item was 34% after 28 days and 35% after 60 days, based on CO2 production. Primary biodegradation of the monoester fraction reached 74% after 7 days, 89% after 28 days and 95% after 60 days.

Additional analysis of extracts from the biodegradation study was conducted in order to identify degradation products (Noack Laboratorien 2017b). Resin acids are early metabolites in the primary degradation of the mono ester fraction of the test item, as demonstrated by an increase in these acids (based on analysis of hydrogenated resin acids and dehydroabietic acid) observed in parallel to a reduction of the mono ester fraction.

The test item is not readily biodegradable based on the ultimate degradation determined in the study. Nevertheless, the test item reveals a considerable potential for biodegradation, as the determination of the primary degradation of the monoester fraction as well as the determination of mineralisation shows.

Description of key information

In order to assess the degradation potential of monoester constituents, a ready biodegradation study was conducted with a test item enriched in monoesters (70.7%). The test item was not readily biodegradable (34% ultimate degradation after 28 days). However, primary degradation of monoester constituents was high (89% after 28 days, 95% after 60 days) and therefore the monoester fraction of the registered substance is not considered to be persistent or very persistent.

Key value for chemical safety assessment

Additional information

The ready biodegradability of the test item Monoesters of hydrogenated rosin with glycerol was determined with a mixed inoculum of the aqueous phase of non-adapted activated sludge and pre-treated, non-adapted standard soil in the Headspace Test following OECD 310 (Noack Laboratorien 2017a). Biodegradation was assessed after 28 days, and the test was then extended to 60 days. Mineralisation of the test item was followed by TIC analyses to assess CO2 production. Primary degradation of the monoester fraction was determined by GC-MS analysis of representative monoester constituents.

The study was conducted using a nominal test item concentration of 33 mg/L, corresponding to a carbon content of 24.6 mg C/L. A stock solution of the test item in acetone was prepared and introduced to the test vessel by pipetting the stock solution around the wall of the test vessel to produce a big surface area. The acetone was allowed to evaporate over 3 days before the test start. CO2 was captured by sodium hydroxide solution and carbon concentrations were determined with a carbon analyser. The test included a reference item control, toxicity control, solvent control and inoculum control. All validity criteria for the study were met. This study is considered to be reliable without restriction (Klimisch 1).

The ultimate biodegradation of the test item was 34% after 28 days and 35% after 60 days, based on CO2 production. Primary biodegradation of the monoester fraction reached 74% after 7 days, 89% after 28 days and 95% after 60 days.

Additional analysis of extracts from the biodegradation study was conducted in order to identify degradation products (Noack Laboratorien 2017b). Resin acids are early metabolites in the primary degradation of the mono ester fraction of the test item, as demonstrated by an increase in these acids (based on analysis of hydrogenated resin acids and dehydroabietic acid) observed in parallel to a reduction of the mono ester fraction.

The test item is not readily biodegradable based on the ultimate degradation determined in the study. Nevertheless, the test item reveals a considerable potential for biodegradation, as the determination of the primary degradation of the monoester fraction as well as the determination of mineralisation shows.